Nonhomologous end-joining (NHEJ) is a newly appreciated pathway of double strand break repair in bacteria. NHEJ operates in a subset of bacteria, including M. tuberculosis and M. smegmatis, that encode the core NHEJ components: the DNA end-binding protein Ku and ATP- dependent DNA ligase D (LigD). In studies supported by this award, we have characterized the NHEJ pathway in M. smegmatis and M. tuberculosis and demonstrated its dependence on Ku and LigD, with a backup role for ATP-dependent DNA ligase C (LigC). We have shown that repair of 5' overhang and blunt-end double strand breaks (DSBs) by NHEJ is highly mutagenic through the activity of the LigD polymerase domain (LigD-POL), a novel bacterial polymerase that also plays a key structural role in the NHEJ complex. We have developed the homing endonuclease I-SceI for cleavage of the mycobacterial chromosome and shown that NHEJ is required for repair of chromosomal DSBs, a process which also introduces insertions and deletions at repaired ends. Building on this foundation, we now propose an expanded program of biochemical and genetic investigation of the mycobacterial NHEJ pathway, its relationship to other pathways of DSB repair, and its role in M. tuberculosis pathogenesis. By using a newly developed assay of chromosomal DSB repair that discriminates HR, NHEJ and single-strand annealing (SSA) pathways, we will determine the relative frequency of pathway use, molecular outcomes, and effects of DSB end-configuration on DSB repair in wild-type M. smegmatis and mutants deficient in NHEJ components, HR components, or both. Prompted by our findings that UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3'-to-5' DNA helicase, we will probe the role of UvrD1 in DNA repair. We will determine the contribution of RecBCD and the novel mycobacterial helicase/nuclease AdnAB to DNA repair and NHEJ deletion formation through a detailed genetic and biochemical analysis of these enzyme complexes. Finally, we will test whether NHEJ and HR play overlapping roles in M. tuberculosis persistence and latency in the murine model. These studies will provide mechanistic insight into prokaryotic NHEJ and determine the role of NHEJ in pathogenesis, potentially advancing this pathway as a target for antimicrobial development.